When tissues (normal and neoplastic) increase in size, they require the formation of microcapillaries (angiogenesis) to provide nourishment to sustain their growth. Constituents of these microcapillaries include most prominently endothelial cells, but also associated mesenchymal cells, fibroblasts, smooth muscle cells, and pericytes. Angiogenesis is important in normal processes, such as wound healing, but also in diseases such as cancer, psoriasis, diabetes, rheumatoid arthritis, and age-related macular degeneration.
There is a need for improved methods for studying microcapillaries in vitro in both normal and diseased tissues. A summary of presently known methods is provided in Staton, et al., “Current Methods for Assaying Angiogenesis in vitro and in vivo,” Int J Exp Path (2004) 85:233-248. In vivo models are useful but cumbersome. In vitro models are less cumbersome but also more artificial and less relevant.
In particular, there is a need for improved methods to predict the activity of anti-cancer drugs and other treatments which target the microvasculature of tumors. For example, bevacizumab (Avastin®) is an FDA-approved anti-cancer drug which targets the microvasculature of tumors. The wholesale cost of Avastin® is more than $40,000 for 10 months of treatment; yet only a relatively small percentage of patients derive substantial benefit. As stated by Ince, et al., “Association of k-ras, b-raf, and p53 Status with the Treatment Effect of Bevacizumab,” J Natl Cancer Inst (2005) 97:981-989, the identification of biomarkers that may predict which patients are most likely to respond to such treatment is of considerable interest.
The most commonly used in vitro methods involve isolating and culturing endothelial cells. Once the cells have been cultured, the effect of drugs (or other perturbations) may be studied, using a variety of cell proliferation and/or cell death endpoints. Examples of cell proliferation endpoints include radioactive thymidine incorporation, cell counting, BrdU incorporation, and colony formation. Examples of cell death endpoints include measurement of cellular ATP, mitochondrial reduction of MTT, metabolism and intracellular trapping of fluorescein diacetate (and loss thereof), loss of cell membrane integrity by dye exclusion, and more specific measurements of apoptosis, such as TUNEL assay or caspase expression. In some cases, previously-isolated endothelial cells have been co-cultured with previously-isolated other cells, and differential effects of drugs on the different cell populations have been studied.
Other in vitro methods are based on organ cultures. For example, see Staton, et al., supra). These include rat aortic ring, chick aortic arch, porcine carotid artery, placental vein disk, and fetal mouse bone explant.
Non-cell culture, non-organ culture, approaches to studying and predicting the effects of bevacizumab have been disclosed by Ince, et al., J Natl Cancer Inst (2005) supra. Ince attempted to correlate k-ras, b-raf, and p53 status with treatment effect of bevacizumab, but concluded that they “did not identify any subgroup of metastatic colorectal cancer patients who were more likely to respond to bevacizumab therapy.” In their discussion, Ince, et al., noted that “To date, few studies have assessed the potential utility of biomarkers in predicting which patients are more likely to respond to antiangiogenic therapy in the clinic” and that no markers had been yet found to be predictive of clinical benefit. These authors suggested that “biomarkers which summarize the effects of all angiogenic regulators may better predict patient outcome than the analysis of a single growth factor or signal induction pathway,” but did not suggest any in vitro methods for this purpose. Instead, they noted ongoing work in which patients themselves are used as experimental models for predicting their own outcomes.
In these studies bevacizumab (and/or other treatments) are administered to the patient on a trial basis and then “early” treatment effects are assessed by means of external diagnostic scanning (e.g., MRI) and/or post-treatment tumor biopsies, with histopathologic evaluation of treatment effects (e.g., Willett, et al., Nature Med (2004) 10:145-147. This approach has many obvious disadvantages, including expense of treatment, exposure of patient to potential toxicity of ultimately ineffective therapy, and the expense of diagnostic studies (e.g., MRI). Such studies also lack of ability to test multiple different treatments simultaneously without risk to the patient as is possible with in vitro methods.
PCT publication WO2007/075440, the work of the applicant herein, describes assessing the effects of treatment on mixtures or microaggregates of endothelial and non-endothelial cells using microscopic observation of absorption of dyes that are rejected by viable cells but taken up by non-viable cells, in particular fast green. The observation was made by the applicant that dead endothelial cells were distinguishable through their appearance from both live and dead tumor cells or non-endothelial cells. This distinction, however, relied on observations that were more readily perceived if the non-endothelial cells themselves were not killed by whatever treatment was administered. In that case, a second indicator dye which is taken up by living cells could be used to contrast living cells from the dead endothelial cells resulting in what was characterized as a blueberry pancake where the dead endothelial cells showed up as “blueberries” against a pink background. Other publications describing this in general, also representing the work of the applicant, include Weisenthal, L. M., et al., J. Intern. Med. (2008) 264:275-287; Weisenthal, L., et al., ASCO 2008 Breast Cancer Symposium, Washington, D.C., Abstract No. 166; and Weisenthal, L, et al., J. Clin. Oncol. (2010) 28:Supplement:Abstract E13617.
Even if the non-endothelial cells were killed by the treatment, a distinction could still be made. Only endothelial cells that die have a distinct appearance being refractile, hyperchromatic and blue-black in appearance when stained with Fast Green, whereas any dead non-endothelial cells were a paler blue. In the subsequent PCT publication, also the work of the current applicant, publication number WO2009/143478, advantage was taken of the distinctive appearance of endothelial cells in response to toxic agents specific therefor to detect and quantify circulating endothelial cells as an index of well being. Again, the appearance of these dead endothelial cells permitted this assessment. Using this method, it was also found that sub-toxic blood levels of ethanol and/or DMSO were useful adjuvants to treatment of unwanted neovasculature.
Although the methods described in the above-referenced PCT publications are effective, they are difficult to adapt to high throughput formats since the contrast between endothelial cells and non-endothelial cells is more intense if the non-endothelial cells are not affected by the treatment. The present invention solves this problem by providing a method whereby the endothelial cells that have been negatively affected by a treatment are readily distinguishable from non-endothelial cells, whether or not the non-endothelial cells have been negatively affected themselves. In addition, it has been found that certain agents effect a particular type of cell death on endothelial cells, whereas non-specific endothelial cell death could alto be effected by different, non-specific agents.
The present method relies on use of dyes that are sensitive to calcium ion. The association of calcium ion with endothelial cell death, in particular in the context of endothelial cells associated with tumors, makes possible the new technique. Calcium accumulation has been observed in the cardiovascular system in the past, but not specifically associated with a specific type of endothelial cell death. For example, Spyridopoulos, I., et al., Arterioscler. Thromb. Vasc. Biol. (2001) 21:439-444 report that oxysterol-induced apoptosis in human endothelial cells is enhanced by alcohol in a calcium dependent mechanism; blockage of calcium influx abrogated the alcohol-mediated enhancement of this toxicity. It is also known that oxidized LDL's induce massive apoptosis of cultured human endothelial cells in a pathway that is calcium dependent. Inhibition of calcium influx resulted in blocking apoptosis (Escargueil-Blanc, I., et al., Arterioscler. Thromb. Vasc. Biol. (1997) 17:331-339). Calcification of cardiac valves is also noted as a pathology in Mohler, E. R., et al., J. Heart Valve Dis. (1999) 8:254-260. On the other hand, alternative mechanisms for oxified LDL apoptosis are described by Harada-Shiba, M., et al., J. Biol. Chem. (1998) 273:9681-9687.
A review article regarding the relationship of calcium channel blockers to apoptosis in cancer was published by Mason, R. P., J. Am. Col. Cardiol. (1999) 34:1857-1866. This article states that both increases and decreases in cellular calcium levels have been shown to promote apoptotic cell death. The role of calcium channel blockers and promoting cancer was adjudged uncertain. A review of endothelial apoptosis is also found in the article by Stefanec, T., Chest (2000) 117:841-854. This review lists increased intracellular calcium ion concentration as a pro-apoptotic stimulus for endothelial cells.
In summary, short term calcium accumulation has previously been associated with endothelial apoptosis wherein calcium has been viewed as a messenger in molecular pathways leading to what is observed as non-specific cell death in endothelial cells, as opposed to its being a central pathogenic agent in and of itself. See, for example, Orrenius, S. et al., Nat Rev Mol Cell Biol (2003) 4:552-565. It has now been found that the calcium itself may act as a pathogenic agent resulting in dead cells with massive accumulation of calcium and having a crystalline appearance permitting ease of detection and distinction from non-specific cell death.
Thus, there has been no understanding in the art that death of endothelial cells can be associated with a massive rise in intracellular calcium ion. This type of cell death, massive calcium accumulation death (MCAD) is the subject of the present application. This was reported by applicant in the publication Weisenthal, L., et al., Nature Proceedings (2010) at HDL.handle.net/10101/npre.2010.4499.1.